Watermelon mosaic virus (species Potyvirus citrulli, WMV) poses a significant threat to various cucurbit species worldwide. In Poland, the virus has been consistently recorded in Cucurbita pepo var. giromontiina crops since 2008. To analyse the genetic diversity and population structure of WMV in Poland, comprehensive surveys were conducted in two major agricultural regions, Greater Poland and Kuyavian-Pomeranian Voivodeships (west-central and mid-northern regions of Poland, respectively). To better understand the WMV occurrence, over 1,800 leaf samples were randomly collected from symptomatic and asymptomatic zucchini and weed plants. Moreover, aphids were also analysed for the presence of the virus. In this study, 154 complete coat protein gene sequences of WMV isolates collected between 2013 and 2024 were obtained using Sanger or high-throughput sequencing. The sequence identity of the Polish WMV isolates ranged from 92.8 to 100% and 95.4 to 100% for nucleotide and amino acid sequences, respectively. Unique mutations were identified in the isolates originating from the Kuyavian-Pomeranian region. Moreover, a novel KEET motif was identified in nine isolates, which has not been reported in previous studies. Most CP codons undergo negative selection; however, eight positively selected sites were detected. Phylogenetic analysis revealed that the Polish isolates were grouped in both classical and emergent main phylogenetic clades. The population analyses indicated limited or no gene flow between WMV subpopulations, suggesting that genetic drift may be considered an important evolutionary force shaping the viral diversity.

Orthotospovirus iridimaculaflavi (iris yellow spot orthotospovirus, IYSV) is one of the most important viral pathogens affecting onions worldwide. The virus is predominantly transmitted by onion thrips and can cause significant losses in cultivated fields. In this study, the identification and molecular characterisation of IYSV isolates originating from onion crops in Poland were carried out. Symptomatic onion plants (Allium cepa L.) were observed in Kujawsko-Pomorskie, Lubelskie, and Wielkopolskie regions of Poland. The plants were characterised by various disease symptoms from mild to severe, resulting in deformation and growth reduction. A total of 40 onion samples were collected, and the presence of IYSV was confirmed in three of them. Moreover, the occurrence of Potyvirus cepae (onion yellow dwarf virus, OYDV) in single and mixed infections with IYSV was observed. The sequences of IYSV obtained in this study were compared with the sequences retrieved from the GenBank database, and the phylogenetic analysis was subsequently conducted. The maximum-likelihood reconstruction revealed that the Polish isolates mainly grouped with isolates originating from Serbia. To our knowledge, it is the first report of IYSV infecting onions in Poland.

Viruses in legume crops constitute one of the main biotic stresses affecting their production worldwide. Currently, more than 160 viruses are known to infect legumes, with most being transmitted by aphids. Moreover, these viruses often occur in mixed infections, the epidemiological significance of which is poorly understood. In this study, monitoring of legume crops in Poland was carried out in 2020, 2023 and 2024 growing seasons. Over 100 plants, with varying disease symptoms, were collected from various locations. From the collected plants, 12 pooled samples were selected for RNAseq, comprising 10 pea (Pisum sativum) samples, one faba bean (Vicia faba var. minor) sample and one lupin (Lupinus albus) sample. We detected the presence of numerous viruses in mixed infections in these Polish legumes. Moreover, in samples from P. sativum and V. faba var. minor collected in 2024, we detected the presence of pea necrotic yellow dwarf virus (PNYDV) and clover yellow vein virus (ClYVV), posing a threat to legume cultivation in Europe. The results of the phylogenetic analysis revealed that the two Polish isolates of PNYDV were diverse and grouped with the isolates from the Czech Republic, Austria and Denmark. Moreover, we also observed the widespread presence of pea enation mosaic virus 1 (PEMV1), pea enation mosaic virus 2 (PEMV2) and pea seed-borne mosaic virus (PSbMV) in the majority of tested samples. These results suggest that the spread of viruses, primarily transmitted by aphids, could have far-reaching consequences for food production and ecosystem functioning.

Phloem, an exceptional plant vascular tissue, facilitates the transport of photoassimilates, RNAs, and other signaling substances from the leaves to the roots throughout the plant. Among the specialized phloem cells are the conductive sieve elements (SEs), which are unique in that they remain alive despite lacking several cell organelles, including the nucleus, plastids, and most mitochondria. These SEs contain a specific proteinaceous structure composed of phloem-specific proteins (P-proteins), whose function is not yet fully understood. Various P-proteins have been characterized in broad range of model species, including Arabidopsis thaliana, and reported in Fabaceae and Cucurbitaceae plants. To date, only one P-protein has been identified in the model tree species Populus trichocarpa. Given the presence of multiple P-protein encoding genes across numerous plant species, we hypothesized the existence of multiple such genes in the Populus genome. Our genomic analysis uncovered 12 genes being potential orthologues to one of A. thaliana P-protein – SEOR (sieve element occlusion-related) genes, which may contribute to the proteinaceous structures observed in differentiating sieve elements. Our transcriptomic and proteomic analyses confirmed the expression of at least seven of these genes, indicating that the protein structure visible in mature sieve elements in P. trichocarpa may be heterogeneous.

Pepino mosaic virus (PepMV) is a highly infectious potexvirus that poses a significant threat to tomato cultivation in greenhouses worldwide. The threat posed by this virus is attributed to by its genetic complexity, characterized by the presence of multiple genotypes in circulation, mixed infections, and ongoing genotype turnover. Surveys of wild Solanum species have identified promising sources of resistance; however, this resistance is often incomplete, manifesting as symptomless, yet virus-positive, plants. When resistance is identified, introgressing of these traits into elite backgrounds is frequently impeded by reproductive barriers and linkage drag. Consequently, there are currently no commercially available cultivars with durable resistance to PepMV. Current control measures rely on stringent hygiene practices, seed health protocols, and the use of mild isolate cross-protection, which can mitigate fruit symptoms when carefully genotype-matched and closely monitored. Looking forward, achieving durable control will likely require host-centered strategies. Loss-of-susceptibility mutations and RNA interference-based approaches have demonstrated strong potential in experimental studies. Future solutions may involve the integration of genome editing with RNA-based technologies, supported by regulatory harmonization and socioeconomic viability considerations.

Background and Aims The transformation of sieve elements from meristematic cells, equipped with a full complement of organelles, to specialized transport tubes devoid of a nucleus has long been enigmatic. We hypothesized a strong involvement of various degradation pathways, particularly macroautophagy in this context, emphasizing the importance of autophagic selectivity in the remaining viability of these cells. Methods Experiments were performed on pioneer roots of Populus trichocarpa cultivated in rhizotrons under field conditions. Through anatomical, ultrastructural and molecular analyses, we delineate the stages of phloemogenesis and the concurrent alterations in the cytoplasmic composition of SEs. Key Results Notably, we observed not only macroautophagic structures, but also the formation of autophagic plastids, the selective degradation of specific organelles, vacuole disruption and the release of vacuolar contents. These events initially lead to localized reductions in cytoplasm density, but the organelle-rich cytoplasmic phase is safeguarded from extensive damage by a membrane system derived from the endoplasmic reticulum. The sieve element ultimately develops into a conduit containing electron-translucent cytoplasm. Eventually, the mature sieve element is a tube filled only by translucent cytoplasm, with sparse organelles tethered to the cell wall. Conclusions Although the activation of programmed cell death pathways was postulated, the persistence of sieve elements indicates that protoplast depletion is meticulously regulated by hitherto unidentified mechanisms. This research elucidates the sequential processes occurring in these cells during phloemogenesis and unveils novel insights into the mechanisms of selective autophagy.

Viral diseases are one of the main factors limiting the cultivation of Capsicum annuum L. worldwide, causing yield losses of up to 80%. Cucumber mosaic virus (CMV) is a prevalent virus affecting the quality and yield of pepper; however, the presence of other viruses has been also noticed. In this study, pepper fruits originated from retail chains, home gardens and greenhouses were analysed to determine the occurrence of viruses infecting sweet pepper crops in Poland. A total of 32 symptomatic and asymptomatic fruit samples were tested for the presence of: CMV, pepper mild mottle virus (PMMoV), tomato spotted wilt virus (TSWV), tomato mosaic virus (ToMV), tobacco mosaic virus (TMV) and potato virus Y (PVY). The presence of viruses in single and mixed infections, with a predominance of CMV, was confirmed. In addition, two symptomatic pepper samples that were negative in RT-PCR for common viruses were subjected to high-throughput sequencing and the presence of new viruses in Poland: bell pepper alphaendornavirus (BPEV) and pepper cryptic virus 2 (PCV2) in a mixed infection with other viruses was demonstrated. The presence of new viruses was verified in all tested samples and BPEV was the most prevalent. The phylogenetic analysis of the BPEV population showed that the Polish isolates are diverse and grouped within two clusters. Although BPEV does not pose a direct threat to pepper crops, its presence may cause changes in the physiology of the host, as well as may interact with other viruses in mixed infections.

Defective viral genomes (DVGs) emerge during error-prone replication of viral genomes and contain deletions, insertions, genomic rearrangements, and hypermutations. These large-effect mutations result in the inability of DVGs to complete an infectious cycle in the absence of a helper wild-type virus. It has been shown that in vitro DVGs usually accumulate in viral populations when a virus is serially passaged in the same host at a high multiplicity of infection. To investigate the impact of host-to- host transmission on DVG formation and population dynamics in vivo, we conducted evolution experiments with tomato black ring virus (TBRV). TBRV was sequentially passaged through a combination of four distinct host species: quinoa, tobacco, lettuce, and spinach. The host was changed every fifth passage. The diversity and popula tion dynamics of DVGs were analyzed based on the RNA-Seq data obtained through sequencing of viral RNA after 20 passages. Our findings indicate the possibility of TBRV DVGs generation when the virus was passaged through different host species. The level of DVG abundance varied across host plant combinations, with a weak indication that the host species past sequence may play a role in DVGs generation. Most abundant DVGs in the TBRV evolved populations were derived from RNA1. Deletions were the most prevalent class of DVGs, followed by insertions. The deletion DVG subpopulation exhibited substantial diversity in species composition and the richness of the deletions species was correlated with their abundance. Longer DVGs characterized by small deletions were predominant, whereas those shorter than 1,000 nucleotides constituted less than 2%.

Tomato black ring virus (TBRV) is a member of the Nepovirus genus in the Secoviridae family, which infects a wide range of important crop species worldwide. In this work, we constructed four cDNA infectious clones of the TBRV tagged with the green fluorescent protein (TBRV-GFP), which varied in (i) the length of the sequences flanking the GFP insert, (ii) the position of the GFP insert within the RNA2 polyprotein, and (iii) the addition of a self-cutting 2A protein. The presence of the GFP coding sequence in infected plants was verified by RT-PCR, while the infectivity and stability of the constructs were verified by mechanical inoculation of the host plants. The systemic spread of TBRV-GFP within plants was observed under UV light at a macroscopic level, monitoring GFP-derived fluorescence in leaves, and at a microscopic level using confocal microscopy. The obtained clones are a valuable tool for future studies of TBRV-host interactions, virus biology, and the long-term monitoring of its distribution in infected plants.

Black locust (Robinia pseudoacacia L.) is characterized by fast growth, low environmental requirements and aesthetic values and therefore it has become a very popular tree planted in forest and urban areas. In recent years, altered shape trees with virus-like symptoms on their leaves have been observed. Therefore, the aim of this work was to identify the virus species potentially causing visible symptoms. During the surveys carried out in 2016–2019, 683 black locust samples were collected. Out of 223 identified virus isolates peanut stunt virus (PSV) (58.7%) was predominant, followed by strawberry latent ringspot virus (SLRSV) (27.8%) and tomato black ring virus (TBRV) (13.5%). The occurrence of mixed infection was also recorded. Annual observation of infected trees confirms the negative impact of viruses on their growth and general condition, leading in many cases to dieback and felling of trees.

Dasheen mosaic virus (DsMV) is one of the most important viral pathogens of aroids and can cause major economic losses for ornamental crops.Here, we present the detection and molecular characterisation of DsMV isolates originating from Monstera adansonii plants in Poland.Moreover, the genetic variability of DsMV isolates was analyzed based on the coat protein gene (CP) of the Polish and other DsMV isolates described to date.The presence of DsMV was confirmed by transmission electron microscopy (TEM) and reverse transcription polymerase chain reaction (RT-PCR) with specific, diagnostic primers in three out of ten examined plants.To obtain full-length sequences of CP, two pairs of primers were designed and used in the RT-PCR.The specificity of obtained products was confirmed by Sanger sequencing.The obtained sequences of CP were compared with 44 other DsMV sequences retrieved from the GenBank.Analyses revealed that DsMV population is very diverse.The variability of DsMV isolates was confirmed by low sequence identity and pervasive recombination events.The phylogenetic analysis was performed based on 37 non-recombinant CP sequences.The maximum-likelihood reconstruction revealed that the Polish isolates are distinct and grouped separately from other DsMV isolates.Due to the high genetic diversity, detecting the virus could be difficult.Nonetheless disease management relies strongly on a fast and accurate identification of the causal agent.To our knowledge this is the first report of DsMV in Poland.

Viral satellite RNAs (satRNAs) are small subviral particles that are associated with the genomic RNA of a helper virus (HV). Their replication, encapsidation, and movement depend on the HV. In this paper, we performed a global analysis of the satRNAs associated with different isolates of tomato black ring virus (TBRV). We checked the presence of satRNAs in 42 samples infected with TBRV, performed recombination and genetic diversity analyses, and examined the selective pressure affecting the satRNAs population. We identified 18 satRNAs in total that differed in length and the presence of point mutations. Moreover, we observed a strong effect of selection operating upon the satRNA population. We also constructed infectious cDNA clones of satRNA and examined the viral load of different TBRV isolates in the presence and absence of satRNAs, as well as the accumulation of satRNA molecules on infected plants. Our data provide evidence that the presence of satRNAs significantly affects viral load; however, the magnitude of this effect differs among viral isolates and plant hosts. We also showed a positive correlation between the number of viral genomic RNAs (gRNAs) and satRNAs for two analysed TBRV isolates.

Tomato-infecting viruses have been considered as a serious threat to tomato crops in Poland. Therefore, during 2014–2021, 234 tomato samples delivered directly by greenhouse tomato growers to Plant Disease Clinic of IPP-NRI were tested. Eight virus species: pepino mosaic virus (PepMV), tomato yellow ring orthotospovirus (TYRV), tomato spotted wilt orthotospovirus (TSWV), potato virus Y (PVY), cucumber mosaic virus (CMV), tomato black ring virus (TBRV) and tomato mosaic virus (ToMV) were detected in single or mixed infection in 89 samples. The presence of TYRV was established for the first time in Poland in 2014. Since then, its presence has been observed in single and mixed infection with TSWV and CMV. Here, we analysed the genetic variability of TYRV population based on complete nucleocapsid (N) protein gene sequence of 55 TYRV isolates. Maximum-likelihood reconstruction revealed the presence of three distinct, well-supported phylogroups. Moreover, the effect of host species on virus diversity was confirmed. Therefore, RT-LAMP assay was developed for the rapid and efficient detection of TYRV isolates that can be implemented in field and greenhouse conditions.

Wirus mozaiki arbuza (watermelon mosaic virus, WMV) oraz wirus żółtej mozaiki cukinii (zucchini yellow mosaic virus, ZYMV) są groźnymi patogenami infekującymi uprawy roślin dyniowatych na całym świecie. W Polsce występują powszechnie, szczególnie w uprawach cukinii, powodując straty w jakości i ilości plonów. Celem pracy było opracowanie i zoptymalizowanie reakcji dupleks RT-PCR do jednoczesnego wykrywania obu wirusów często stwierdzanych w infekcji mieszanej. Zaprojektowane startery amplifikowały gen kodujący białko płaszcza (CP) obu wirusów odpowiednio wielkości: 977 pz WMV oraz 415 pz ZYMV. Czułość wykrywania reakcji dupleks RT-PCR wyniosła 500 pg/μl całkowitego RNA. Specyficzność wykrywania poszczególnych produktów była sprawdzana za pomocą sekwencjonowania metodą Sangera. opracowana technika może być wykorzystywana do rutynowego wykrywania obu wirusów w zainfekowanych próbkach.

Onion yellow dwarf virus is distributed worldwide significantly reducing yield of crops from the Allium genus. The aim of the study was the detection and molecular characterization of newly identified OYDV isolates infecting onions in Poland. The virus was detected by transmission electron microscopy and RT-PCR techniques using two pairs of diagnostic primers: OYDV-NibCPF1/R1 and OYDV-CPF2/R2. The specificity of obtained RT-PCR products was confirmed by Sanger sequencing and received viral coat protein sequence was used for phylogenetic analysis. The phylogenetic analysis was carried out using CP sequences of the new Polish onion isolate obtained in this study and 37 other sequences of OYDV retrieved from GenBank. The analysis revealed that the Polish OYDV isolate is the most similar to the OYDV isolates derived from onions from Argentina and Germany, which may indicate their common origin. Moreover, it was observed that the Polish onion and garlic isolates are very diverse and belong to different phylogroups.

Wirus mozaiki arbuza (watermelon mosaic virus, WMV) oraz wirus żółtej mozaiki cukinii (zucchini yellow mosaic virus, ZYMV) są groźnymi patogenami infekującymi uprawy roślin dyniowatych na całym świecie. W Polsce występują powszechnie, szczególnie w uprawach cukinii, powodując straty w jakości i ilości plonów. Celem pracy było opracowanie i zoptymalizowanie reakcji dupleks RT-PCR do jednoczesnego wykrywania obu wirusów często stwierdzanych w infekcji mieszanej. Zaprojektowane startery amplifikowały gen kodujący białko płaszcza (CP) obu wirusów odpowiednio wielkości: 977 pz WMV oraz 415 pz ZYMV. Czułość wykrywania reakcji dupleks RT-PCR wyniosła 500 pg/μl całkowitego RNA. Specyficzność wykrywania poszczególnych produktów była sprawdzana za pomocą sekwencjonowania metodą Sangera. Opracowana technika może być wykorzystywana do rutynowego wykrywania obu wirusów w zainfekowanych próbkach. Watermelon mosaic virus (WMV) and zucchini yellow mosaic virus (ZYMV) are dangerous pathogens infecting cucurbit crops worldwide. In Poland, both viruses are common, especially in the cultivation of zucchini, causing losses in the quality and quantity of crops. The aim of the study was to develop and optimize the duplex RT-PCR reaction for simultaneous detection of both viruses often occurring in mixed infection. The designed primers amplified the gene encoding the coat protein (CP) of both viruses with a size of 977 bp for WMV and 415 bp for ZYMV. The duplex RT-PCR detection limit was 500 pg/μl of total RNA. The specificity of detection of individual products was checked by Sanger sequencing. The developed assay can be used for routine detection of both viruses in infected samples.

Beech seeds are produced irregularly, and there is a need for long-term storage of these seeds for forest management practices. Accumulated reactive oxygen species broadly oxidize molecules, including amino acids, such as methionine, thereby contributing to decreased seed viability. Methionine oxidation can be reversed by the activity of methionine sulfoxide reductases (Msrs), which are enzymes involved in the regulation of many developmental processes and stress responses. Two types of Msrs, MsrB1 and MsrB2, were investigated in beech seeds to determine their abundance and localization. MsrB1 and MsrB2 were detected in the cortical cells and the outer area of the vascular cylinder of the embryonic axes as well as in the epidermis and parenchyma cells of cotyledons. The abundances of MsrB1 and MsrB2 decreased during long-term storage. Ultrastructural analyses have demonstrated the accumulation of these proteins in protein storage vacuoles and in the cytoplasm, especially in close proximity to the cell membrane. In silico predictions of possible Msr interactions supported our findings. In this study, we investigate the contribution of MsrB1 and MsrB2 locations in the regulation of seed viability and suggest that MsrB2 is linked with the longevity of beech seeds via association with proper utilization of storage material.

Viruses cause epidemics on all major crops of agronomic importance, and a timely and accurate identification is essential for control. High throughput sequencing (HTS) is a technology that allows the identification of all viruses without prior knowledge on the targeted pathogens. In this paper, we used HTS technique for the detection and identification of different viral species occurring in single and mixed infections in plants in Poland. We analysed various host plants representing different families. Within the 20 tested samples, we identified a total of 13 different virus species, including those whose presence has not been reported in Poland before: clover yellow mosaic virus (ClYMV) and melandrium yellow fleck virus (MYFV). Due to this new finding, the obtained sequences were compared with others retrieved from GenBank. In addition, cucurbit aphid-borne yellows virus (CABYV) was also detected, and due to the recent occurrence of this virus in Poland, a phylogenetic analysis of these new isolates was performed. The analysis revealed that CABYV population is highly diverse and the Polish isolates of CABYV belong to two different phylogenetic groups. Our results showed that HTS-based technology is a valuable diagnostic tool for the identification of different virus species originating from variable hosts, and can provide rapid information about the spectrum of plant viruses previously not detected in a region.

Pepino mosaic virus (PepMV) is a highly infectious virus causing significant losses in tomato greenhouse production worldwide. One of the characteristic symptoms is yellowing discolorations of leaf blades, which is often accompanied by damage of the fruits. It has been found that, two independent point mutations in the gene encoding the coat protein (CP) are responsible for the formation of yellowing symptoms: G463AA/A463AA (155 aa) and GA497U/GG497U (166 aa), respectively. In our study, qPCR-HRM was used to distinguish mild and yellowing pathotypes. Appropriate primers, amplifying a fragment of the CP-encoding gene in which both yellowing mutations are located were designed. The analysis was carried out using 18 isolates originated from greenhouses in different region of Poland. Our experiments confirmed the effectiveness of the designed primers to differentiate the analyzed pathotypes. This technique is a valuable tool for detecting the diversity of PepMV. Wirus mozaiki pepino (PepMV) jest groźnym patogenem powodującym znaczne straty w uprawie szklarniowej pomidora w wielu regionach świata. Jednym z charakterystycznych objawów obecności wirusa w roślinie są żółtaczkowe przebarwienia blaszek, którym często towarzyszy uszkodzenie owoców. Za ich powstawanie odpowiedzialne są dwie niezależne mutacje punktowe w genie kodującym białko płaszcza (CP): G463AA/A463AA (155 aa) i GA497U/GG497U (166 aa). W przeprowadzonych badaniach wykorzystano qPCR-HRM do rozróżniania łagodnych i żółtaczkowych patotypów. Zaprojektowano startery, amplifikujące fragment genu kodującego CP, w którym znajdują się obie mutacje żółtaczkowe. Przeanalizowano 18 izolatów, pochodzących z obiektów szklarniowych na terenie całego kraju. Wykonane doświadczenia potwierdziły skuteczność zaprojektowanych starterów do różnicowania analizowanych patotypów. Technika ta może być z powodzeniem stosowana do rozróżniania łagodnych i żółtaczkowych patotypów PepMV na terenie naszego kraju.

Tomato black ring virus (TBRV) is a worldwide-distributed RNA virus infecting a wide range of different host plants, including crop species, trees, shrubs, and weeds. Here, we investigated the molecular evolution of TBRV and its adaptability to different plant species. The TBRV-Pi isolate was used to generate five independent evolution lineages serially passaged in either quinoa, tobacco, or tomato plants. After 15 passages, the genetic variability present in all the lineages was characterized for the movement (MP) and coat (CP) coding cistrons. We addressed two main questions: to what extent does the amount of genetic variability in the TBRV genome depend on the host species, and are there host species-specific adaptive mutations? Overall, 201 different nucleotide substitutions emerged during the evolution experiment, with some of them appearing multiple times in different lineages; two of them (one inCPand one inMP) were unique for a particular host plant. We have shown that the degree of genetic differentiation depends on the host species in which the virus evolved, and that positive selection is operating upon certain residues, particularly inCP. Moreover, we have characterized new types of defective RNAs that arose during the TBRV-Pi evolution in tobacco. Furthermore, this is the first report of a defective RNA from the RNA2 of TBRV.